Acetylcholinesterase (AChE) is an important marker for understanding the development, organization and regulation of the neuromuscular synapse. Studies of AChE have provided novel and fundamental information towards understanding how nerves regulate skeletal muscle, and studies of the synaptic collagen-tailed AChE form provided knowledge about the formation of the synaptic basal lamina. Dr. Rotundo and colleagues have now developed several new probes and techniques for studying AChE biogenesis, targeting and regulation that will provide new information about the structure and assembly of this prototypical and accessible synapse. Their specific aims are: (1) to continue transcriptional regulation studies to test the hypothesis that the different AChE transcripts arise from different activity-dependent promoters; to study the activity dependent expression of the synapse-specific collagen-like tail subunit by RNase protection, nuclear run-on, and in situ hybridization; (2) to determine the molecular mechanisms responsible for targeting AChE to the synapse including testing the hypothesis that localized exocytosis occurs using secretable Green Fluorescent Protein-AChE chimeric molecules; determining the organization of AChE and nicotinic receptor molecules on the cell surface using new fluorescent and biotinylated ligands that they have synthesized that reveal new relations between the two types of molecules; and testing the hypothesis that complexes of synaptic basal lamina components including AChE attached to perlecan HSP attached to alpha-dystroglycan are organized intracellularly prior to externalization; and (3) continuing their studies on the second messenger systems involved in transducing membrane depolarization into changes in AChE gene regulation, and their studies indicating the presence of muscarinic receptors in skeletal muscle linked to diacylglycerol production and ultimately regulation of AChE expression at the transcriptional and translational levels. These studies will help better understand how nerves control muscle function at the molecular level, and what happens to muscle when it loses the regulatory influence of the nerves.